In order to investigate how energy expenditure changes with over and underfeeding the following studies are underway. In one study, after careful calibration of weight maintenance EE, individuals undergo a series of measurements of 24 hour EE in a respiratory chamber in which they are fasting or overfed (by 200% of weight maintenance needs) a series of diets that vary in macronutrient content. This is to further investigate whether low or high protein diets may improve the detection of recruitment of adaptive thermogenesis. In addition, behavioral, metabolic and hormonal tests are performed to examine associated characteristics and to investigate the mechanism of the changes in EE. These individuals will also be followed up long term to look at what factors predict weigh change. We found that the change in energy expenditure with fasting and with overfeeding is reproducible. In addition in our initial results, the increase in energy expenditure with overfeeding was highest in those overfed a normal protein high fat or high carbohydrate diet. Energy expenditure did not increase significantly with low protein diets. We have now enrolled over 80 participants in this study. We have confirmed the negative association between the percent change in EE with overfeeding with the percent change in EE with fasting indicating the presence of thrifty versus spendthrift phenotypes. At 6 month follow-up after 6 months we found that those who had less decrease in EE with fasting, and greater increase in EE with low protein diets gained more weight. In addition, higher EE with high carbohydrate overfeeding predicted weight gain. In models with all three EE measures, less EE response to low protein overfeeding and higher EE response to high carbohydrate overfeeding were the main predictors of weight change. Using data from 24 hour urine catecholamines collected during the diets, there is evidence of sympathetic nervous system activation during these diets (but not the others). However, the main increase is in epinephrine (as opposed to norepineprhrine) implicating a role for the adrenal medulla. Based on the difference in energy expenditure measured during fasting and overfeeding, the thermic effect of food (TEF) can be calculated. We have found that TEF is inversely associated with adiposity. Thus TEF may predispose to weight gain or be an adaptation to increased bodyweight. As increased adiposity may insulate against trans-abdominal heat loss which may increase TEF, we are investigating the effect of central insulation on the EE and TEF changes associated with overfeeding. We are also continuing to measure EE with overfeeding and fasting, and investigating whether these changes predict longer term weight gain. Brown fat is thought to be etiologic in diet induced thermogenesis in rodents and is known to be present in adults. We investigated whether brown fat was activated with overfeeding. Using a high fat overfeeding diet (200% of energy balance calories) which produces a sustained increase in EE including during sleep, we did not find any evidence of brown fat activation with overfeeding in humans. However, using a high carbohydrate overfeeding diet we have seen preliminary evidence of brown fat activation in some subjects. We are in the process of preparing a new study that will use PET-MRIs to investigate brown fat activation in subjects exposed to cold and after overfeeding with both a high fat and high carbohydrate diet. In a related study, after measurement of EE with over and underfeeding, and also after undergoing a series of metabolic and behavioral testing (including biopsies of muscle and fat), individuals are admitted for 6 weeks of an inpatient dietary protocol involving underfeeding (for overweight and obese individuals) or overfeeding (for lean, obesity resistant individuals). During the inpatient study, all aspects of food intake, energy expenditure, and energy loss are carefully measured to determine if differences in weight gain or loss can be attributed to recruitment of adaptive thermogenesis or other factors. We have currently completed 12 individuals who have undergone the weight loss study. We found that less decrease in 24hEE with fasting was associated with greater weight loss in this controlled inpatient study. Furthermore using measured energy expenditure and physical activity calories and directly measured calories from consumed food and excreted urine and stool we were able to calculate an accumulated energy deficit. Those with greater accumulated energy deficit also had less decrease in EE with fasting measured prior to the weight loss period, and lost more weight. . These results indicate the presence of spendthrift versus thrifty phenotypes even in obese individuals undergoing weight loss, and accounts in part for the variability in dietary weight loss. In investigating other metabolic features of these thrifty versus spendthrift phenotypes, we have found that more thrifty individuals tend to have more fat mass, and lower core body temperatures. We are continuing to measure related hormones and investigate expression data from fat and muscle to characterize these phenotypes. We are also continuing to enroll lean individuals in the overfeeding arm to assess these phenotypes with weight gain. Recruitment for both studies is ongoing.